This invention relates to the purification of a cancer-associated protein and to the preparation of an antibody thereto.
In spite of improved treatments for certain forms of cancer, it is still the second leading cause of death in the United States. Since the chance for complete remission of cancer is, in most cases, greatly enhanced by early diagnosis, it is very desirable that physicians be able to detect cancers before a substantial tumor develops. Also, in cases where the primary tumor has been substantially removed by surgery or destroyed by other means, it is important that the physician be capable of detecting any trace of cancer in the patient (either in the form of residues of the primary tumor or of secondary tumors caused by metastasis), in order that the physician can prescribe appropriate subsequent treatment, such as chemotherapy.
The quantities of cancer cells that must be detected for early diagnosis or following removal or destruction of the primary tumor are so small that the physician cannot rely upon physical examination of the cancer site; moreover, in many cases the cancer site is of course not susceptible of direct visual observation and it is almost always impractical to detect secondary tumors by visual observation, since it is not possible to predict exactly where they are likely to occur. Accordingly, sensitive tests have to rely upon detection of cancer-associated materials, usually proteins, present in body fluids of patients who have, or are about to develop, small numbers of cancer cells in their bodies. Diagnostic materials for detection of at least two cancer-associated proteins are already available commercially. Tests for alpha-fetoprotein are used to detect primary liver cancer and teratocarcinoma in humans; see, for example:
Abelev, Production of Embryonal Serum Alpha-Globulin by Hepatomas: Review of Experimental and Clinical Data, Cancer Res. 28: 1344-1350 (1968); PA1 Abelev et al, Embryonal Serum Alpha-Globulin in Cancer Patients: Diagnostic Value, Int. J. Cancer, 2: 551-558 (1967); and PA1 Esterhay et al, Serum Alpha-Fetoprotein Concentration and Tumor Growth in a Patient with Ovarian Teratocarcinoma, Cancer (Philadelphia), 31: 835-839 (1973). PA1 Gold and Freedman, Demonstration of Tumor-Specific Antigens in Human Colonic Carcinomata by Immunological Tolerance and Absorption Techniques, J. Exp. Med., 121: 439-462 (1965); and PA1 Terry et al, Carcinoembryonic Antigen: Characterization and Clinical Applications, Transplant Rev., 20: 100-129 (1974). PA1 Schumm et al, Apparent Transformed Cell-dependent Proteins in Blood Plasma, Proc. Am. Assoc. Cancer Res., 22: 79 (1981); PA1 Schumm and Webb, Differential Effect of Plasma Fractions from Normal and Tumor-Bearing Rats on Nuclear RNA Restriction, Nature (London), 256: 508-509 (1975); PA1 Schumm and Webb, Modification of Nuclear Restriction In Vitro by Plasma from Tumor-Bearing Animals, J. Natl. Cancer Inst., 54: 123-128 (1975); and PA1 Schumm and Webb, Apparent Transformed Cell-Dependent Proteins in Blood Plasma, Proc. Am. Assoc. Cancer Res., 22: 79 (1981). PA1 Schumm et al, Changes in Nuclear RNA Transport Incident to Carcinogenesis, Eur. J. Cancer, 13: 139-147 (1977); PA1 Schumm et al, Cytosol-Modulated Transport of Messenger RNA from Isolated Nuclei Cancer Res., 33: 1821-1828 (1973). PA1 (a) not being precipitated by 30% saturated aqueous ammonium sulfate solution at 25.degree. C.; PA1 (b) having a molecular weight of approximately 60,000; PA1 (c) being precipitated from aqueous solution by 3.3% streptomycin sulfate; PA1 (d) having substantially no autophosphorylation activity but being phosphorylated with adenosine triphosphate in the presence of an exogenous protein kinase; PA1 (e) having substantially no protein kinase activity; PA1 (f) having the capacity to liberate ribonucleic acid from cell nuclei; and PA1 (g) being substantially free of albumin. PA1 50 mM Tris-HCl (pH 7.5) PA1 25 mM potassium chloride PA1 2.5 mM magnesium chloride PA1 0.5 mM calcium chloride PA1 0.3 mM manganese chloride PA1 5.0 mM sodium chloride PA1 2.5 mM phosphoenol pyruvate PA1 35 units/milliliter of pyruvate kinase PA1 2.5 mM sodium dihydrogen phosphate PA1 5.0 mM spermidine PA1 2.0 mM dithiothreitol PA1 2.0 mM adenosine triphosphate PA1 300 microgram/milliliter of low molecular weight yeast RNA. PA1 (a) separating from the plasma of a mammal suffering from cancer the fraction of plasma protein which is not precipitated by 30% saturated aqueous ammonium sulfate solution; PA1 (b) dispersing this fraction of plasma protein in a buffer and dialyzing the resultant protein solution against the buffer; PA1 (c) separating the fraction of the dialyzed protein having a molecular weight of about 60,000; and PA1 (d) removing substantially all albumin from the 60,000 molecular weight fraction.
Similarly, carcinoembryonic antigen is used for diagnosis of cancers of the digestive system; see, for example:
Unfortunately, both the aforementioned tests are only applicable to a narrow range of cancer types, and therefore these tests suffer not only from the disadvantage that other types of cancer may be missed but also from the disadvantage that the narrow applicability of the tests means that it may be necessary to run multiple tests on a single patient for diagnostic purposes, a procedure which not only increases the expense of the diagnostic testing but also increases the risk that one or other of the tests may give a false positive result. Accordingly, there is a need for a single chemical test able to detect the presence of very small amounts of cells of a wide variety of different cancers.
It is already known that serum from the blood of animals suffering from a wide variety of cancers contains a protein (hereinafter referred to as "cancer marker protein") having a molecular weight of approximately 60,000 and having the capacity to increase the release of ribonucleic acid (RNA) from cell nuclei; see:
Thus, testing for this cancer marker protein is a potential, sensitive method for the detection of a wide variety of cancers in humans and other mammals. Unfortunately, the only method of detecting this cancer marker protein described in the aforementioned papers involves assaying the ability of serum protein from the patient being tested to release RNA from rat liver nuclei; see:
Although, as shown in the aforementioned papers, this test is capable of detecting cancer in patients, the assaying method used is not suitable for routine use by medical technicians. Accordingly, there is a need for a simpler, less costly test for detection of this cancer marker protein.
One method often used for detection of proteins and other antigens is radioimmunoassay. In this technique, a sample of the material to be assayed is mixed with a known quantity of a radioactive-labeled form of the antigen to be measured. The resultant mixture is then treated with an antibody to the antigen being assayed, thereby forming an antigen-antibody complex. The degree of radioactivity of this complex depends upon the amount of the appropriate antigen in the sample being assayed and thus, by measuring the radioactivity of the complex, the quantity of the antigen in the sample may be determined. Radioimmunossay techniques are suitable for routine use by medical technicians and indeed are already used for assay of a variety of antigens in various body fluids. Thus, radioimmunoassay is potentially an attractive technique for testing for the cancer marker protein. However, a radioimmunoassay technique requires the prior preparation of an antibody which is specific to the antigen being assayed and the aforementioned papers do not describe any method for the preparation of such an antibody. Moreover, to prepare an antibody specific to the cancer marker protein, it is necessary to prepare the cancer marker protein in a relatively pure form, since the antibodies have to be prepared by injecting the cancer marker protein into an animal and recovering serum therefrom, and any impurities in the cancer marker protein may give rise to corresponding antibodies to those impurities, thereby interfering with the accurate assay of the cancer marker protein in the radioimmunoassay test.
Another method for the detection of proteins and other antigens is known as an ELISA assay. In this assay, the walls of wells in a culture plate are coated with an antibody to the antigen to be assayed and the wells are then washed. A specimen of the material to be tested is placed in the coated wells, incubated for a period sufficient to allow any antigen present to react with the antibody on the walls, and after incubation the wells are again washed out. Next, there is added to the wells a conjugate of the antibody and horseradish peroxidase, the plates are incubated and washed out, and a mixture of hydrogen peroxide in a buffer and 2,2'-AzinoDi(3-Ethylbenzthiazoline sulfonic Acid) (ABTS) and the plates are allowed to develop for ten minutes at room temperature. The reaction was then stopped by the addition of 2 mM sodium azide solution. If the antigen being assayed (i.e. that specific to the antibody) is not present in the test material, no color will be formed in the wells of the plates because the peroxidase enzyme will have been washed away. If, however, the antigen being assayed is present in the test material immunological reactions will cause the enzyme to adhere to the walls of the wells and this enzyme will cause formation of a color with the hydrogen peroxide/ABTS mixture. Thus, the formation of color at the end of the test is indicative of the presence of the antigen. Like radioimmunoassay, this assay technique requires the preparation of an antibody specific to the antigen being assayed and the preparation of this antibody requires purification of the antigen.
Accordingly, there is a need for a method of preparing the cancer marker protein in a relatively pure form and a method for producing an appropriate antibody to this cancer marker protein. This invention seeks to meet these requirements.